During the past year, we achieved the following objectives for each of our goals: 1. Use of iPSCs and derived NSCs for phenotyping and drug screening We generated a large collection of iPSCs from individuals with trisomy 21 (T21) and age/sex matched euploid (Eup) controls. Using live cell imaging every 3h, we measured the proliferation rates of T21 (N=11) and Eup (N=11) fibroblasts from independent donors. We transformed these fibroblasts to iPSCs using Sendai virus and imaged the formation of colonies daily. We showed that T21 samples grew slower (area under the curve AUC= 4509754) compared to Eup AUC=5837754. When samples were analyzed in pairs, significant differences in growth rate were observed at 24 and 48h. Similar to fibroblast growth data, we measured a significant variability in colony number between individual T21 samples (p<0.05, F test); however, no statistically significant differences were observed in average colony size over time between T21 and Eup. Our data suggest that inter-individual variability exists even at the cellular level in T21, which implies the need for greater numbers of independent cell lines and novel statistical methods when characterizing phenotypes and responses to treatment. We generated neural stem cells from all iPSC lines and we are currently using transcriptome/proteome analysis, live cell imaging and functional assays to characterize the phenotypic alterations in T21 versus Eup cells. iPSC and matching NSC lines will be used for drug screening using the candidate molecules identified previously using the Connectivity Map database (PMID 27586445). 2. Determine the best mouse model of DS Translating human cognitive testing in mouse models of Down syndrome: Infants with Down syndrome (DS) demonstrate delays motor development and in the acquisition of expressive language. We are investigating these two phenotypes using video-tracking in an open field and ultrasonic vocalization (USV) between postnatal days 2 and 12. Under baseline conditions, Ts65Dn neonates emit fewer total calls during the first postnatal week (p<0.01) compared to Eup. Ts1Cje pups emit similar number of total USVs versus their littermate euploid controls. USV classification analysis shows both Ts65Dn and Ts1Cje pups show significantly shorter USVs (p<0.001) and less complex USVs (flat, up, down, chevron, step up and step double) compared to their Eup littermates (p<0.05). In the open field test, Ts1Cje and Ts65Dn pups travel a significantly smaller distance at a significantly lower average speed than euploid littermates. These data suggest abnormal motor and vocalization development in the Ts1Cje and Ts65Dn mouse models. Analyses of motors development and USVs in the Dp(16)1/Yey and Ts66Yah mouse models are ongoing. In the next fiscal year, we will be investigating cognitive deficits in these mouse models using touch screen CANTAB (Cambridge Neuropsychological Test Automated Battery) paradigms. Investigating brain morphometry and cellularity: Intellectual disability and microcephaly are the hallmarks of DS. Individuals with DS exhibit hypoplasia of the cerebellum, hippocampus and frontal lobe. These abnormalities result from reduced neurogenesis and synaptogenesis and hypomyelination. Brains from individuals with DS also show astrogliosis and increased inflammation. We used Diffusion Tensor Imaging (DTI) to evaluate brain morphometric alterations in the Ts65Dn mouse model. We measured a significant reduction in the cerebellum volume and increased diffusivity while white matter had reduced volume and decreased diffusivity. Analysis of the Ts1Cje and Dp(16)1/Yey mouse brain is ongoing. To confirm DTI findings and correlate morphometric changes with potential cellular abnormalities, we performed stereological analysis in the cerebral cortex, hippocampus and cerebellum using cell specific markers (Nissl for neurons and glia, NeuN for neurons, S100 and GFAP for astrocytes, Olig2 for oligodendrocytes and Iba1 for microglia). In the Ts65Dn mice, Layer IV of the somatosensory cortex was significantly thinner that WT littermates. No changes in the cortical thickness was observed in the Ts1Cje and Dp(16)1/Yey models. Dp(16)1/Yey mice had significantly larger lateral ventricles than their WT littermates but no changes were observed in the Ts65Dn and Ts1Cje models. In the cerebellum, Ts65Dn mice had a significantly thinner molecular layer compared to their WT littermates. No change was observed in the Ts1Cje and Dp(16)1/Yey models. All three mouse models showed normal hippocampal morphometry. Analysis of neuronal, astroglial, oligodendrocytic and microglial cell densities is ongoing. Placental and embryo studies in three mouse models of Down syndrome: Mouse models of DS permit deep phenotyping of embryonic and placental phenotypes, which to date have not received much emphasis. We analyzed E18.5 embryos by a veterinary pathologist blinded to genotype for the presence of congenital and placental anomalies. Embryos were characterized as euploid (E), trisomic, mild (TM), or trisomic, severe phenotype (TS) using embryo weight (mild = euploid median, severe = <10th percentile for euploid median). Euploid embryos showed no anomalies; 1 was demised. Hepatic necrosis was seen in Dp(16)1/Yey TM (n=1), TS (n=3) and Ts1Cje embryos TM (n=1); hepatic congestion/inflammation was observed in Ts65Dn embryos TM (n=2), TS (n=1). Other anomalies noted in Ts1Cje include ventricular septal defects TM (n=1), TS (n=1) and brain necrosis TS (n=2). In Ts65Dn embryos 1 had an overriding aorta TS and 1 had hydronephrosis TM. In Ts65Dn placentas, there was increased syncytiotrophoblast necrosis and absence of giant cells in trisomic compared to euploid. No differences were seen in Dp(16)1/Yey or Ts1Cje placentas. The presence of liver abnormalities in all three mouse models of Down syndrome (8/34) is a novel finding. Future research will examine autopsy material to determine if this finding is relevant to humans with Down syndrome. Apigenin as first proof of principle molecule We tested the safety and efficacy of apigenin, a candidate therapy previously identified by uploading gene expression profiles from nine different tissues (six human, three mouse) into the Connectivity Map www.broadinstitute.org/connectivity-map-cmap. We cultured human amniocytes from living human fetuses with T21 and euploid karyotypes for three days with apigenin (0-4M). We analyzed the effects of apigenin on oxidative stress and antioxidant capacity. Apigenin treatment (333-400 mg/kg/day), mixed with chow, was initiated prenatally to Ts1Cje dams and fed to the pups over their entire lifetimes. We investigated the effects of apigenin on fetal brain gene expression, neonatal and adult behavior. In vitro, apigenin significantly reduced oxidative stress and improved antioxidant defense response in T21 cells. In the Ts1Cje mouse model, there was no increase in birth defects or pup deaths resulting from antenatal apigenin treatment. In Ts1Cje neonates, apigenin significantly improved several developmental milestones and particularly, spatial olfactory memory. In addition, in adult mice we noted sex-specific effects on exploratory behavior and long-term hippocampal memory, with males showing significantly more improvement than females. Global gene expression analyses demonstrated that apigenin targets similar signaling pathways (G2/M cell cycle transition, G-protein signaling and NFB signaling) through common upstream regulators (PTGER2, HGF, IFNGR, IKBKB and Forkhead transcription factors) both in vitro and in vivo. Validation of this mechanism of action using live cell imaging is ongoing. These studies provide proof-of-principle that apigenin has therapeutic effects in preclinical models of Down syndrome.